Anticancer, photoluminescence and electrochemical properties of structurally characterized two imine derivatives

Article abstract of DOI:10.1016/j.saa.2015.05.021

Two imine compounds, 4-[(E)-(2-methoxybenzylidene)amino]phenol (L¹) and 4-[(E)-(3,4-dimethoxybenzylidene)amino]phenol (L²) were synthesized and characterized by the analytical and spectroscopic methods. The electrochemical and photol

Full text of DOI:10.1016/j.saa.2015.05.021

Accepted Manuscript
Anticancer, photoluminescence and electrochemical properties of structurally
characterised two imine derivatives
Gökhan Ceyhan, Muhammet Köse, Mehmet Tümer, İbrahim Demirtaş
PII:
S1386-1425(15)00609-5
http://dx.doi.org/10.1016/j.saa.2015.05.021
SAA 13688
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomo-
lecular Spectroscopy
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Received Date:
Revised Date:
Accepted Date:
8 December 2014
17 March 2015
5 May 2015
Please cite this article as: G. Ceyhan, M. Köse, M. Tümer, İ. Demirtaş, Anticancer, photoluminescence and
electrochemical properties of structurally characterised two imine derivatives, Spectrochimica Acta Part A:
Molecular and Biomolecular Spectroscopy (2015), doi: http://dx.doi.org/10.1016/j.saa.2015.05.021
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Anticancer, photoluminescence and electrochemical properties of
structurally characterised two imine derivatives
Gökhan Ceyhana, Muhammet Kösea, Mehmet Tümera*, Đbrahim Demirtaꢀb
aChemistry Department, K.Maras Sütcü Imam University, 46100, K.Maras, Turkey.
bChemistry Department, Çankırı Karatekin University, 18100, Çankırı, Turkey.
ABSTRACT
Two imine compounds, 4-[(E)-(2-methoxybenzylidene)amino]phenol (L1) and 4-[(E)-(3,4-
dimethoxybenzylidene)amino]phenol (L2) were synthesized and characterized by the
analytical and spectroscopic methods. The electrochemical and photoluminescence properties
of the imine compounds L1 and L2 were investigated in different solvents. The compounds L1
and L2 show different redox processes at some potentials. The molecular structures of two
Schiff base compounds are broadly similar, differing principally in the position, the number of
metoxy(-OCH3) groups and dihedral angles between aromatic rings. While the compound L1
has only one metoxy group located on the o-position with respect to the imine bond (C=N),
the L2 contains two metoxy groups on the p-m-positions with respect to the imine bond. The
imine compounds show two or three emission bands in the 619-832 nm range in organic
solvents. In the 1.0 x 10-3 M concentration, the compounds have the highest excitation and
emission bands. The imine compounds L1 and L2 were screened for their in vitro cytotoxicity
on HeLa cell lines using the xCELLigence system (Real Time Cell Analyzer).
Keywords: Schiff base, Anticancer, Electrochemistry, Photoluminescence
*Corresponding author: Mehmet Tümer
Tel.: +90 344 280 14 44, Fax: +90 344 280 13 52,
E-mail address: mtumer@ksu.edu.tr
1

1. Introduction
Imine compounds (called also Schiff bases) are synthesized from the reaction of
aliphatic or aromatic primary amines with aldehydes or ketones. They are nitrogen analogue
of an aldehyde or a ketone in which the carbonyl group (CO) has been replaced by an imine
(azomethine -C=N-) group. Schiff bases form an important class of the most widely used as
organic compounds and has a wide variety of applications in many fields, e.g., biological,
inorganic and analytical chemistry. Some of them are the basic units in certain dyes and are
also used as liquid crystals. Schiff bases appear to be important intermediates in a number of
enzymatic reactions involving interaction of an enzyme with an amino or a carbonyl group of
a substrate [1].
Imine compounds, obtained frequently from various of the heterocyclic primary
amines, were reported to possess a broad spectrum of pharmacological activities with a wide
range of biological properties, development of a new chemotherapeutic imine compounds is
now attracting the attention of medicinal chemists [2]. They are known to exhibit a wide
variety of potent activities. The pharmacologically useful activities include antibacterial,
anticonvulsant, antiinflammatory, anticancer, anti-hypertensive, anti-fungal, antipyretic,
antimicrobial, anti-HIV, cytotoxic activity, hypnotic and herbicidal activities [3].
Our research group reported a series of imine compounds and their transition metal
complexes as biocidal against bacteria and fungi species [4-10]. The biological effects of
these compounds were found to be dose dependent. In our other research, the polydentate
Schiff base ligands and their Cu(II) and Cd(II) complexes were synthesised [11]. In this
research, the Schiff base complexes of cadmium were found to inhibit the intense
chemiluminescence reaction in dimethylsulfoxide (DMSO) solution between luminol and
dioxygen in the presence of a strong base. This effect is significantly correlated with the
stability constants KCdL of the complexes and the protonation constants KOH of the ligands; it
also has no significant association with antibacterial activity. We investigated the antioxidant
and anticancer properties of the sterically hindered Schiff base compounds [12-14]. Higher
absorbance of the reaction mixture indicated greater reducing power of the Schiff base
ligands.
In this study, two imine compounds from the reaction of the 2-methoxy benzaldehyde
or 3,4-dimethoxy benzaldehyde with 4-aminophenol in the ethanolic solution were prepared
and characterized by the analytical and spectroscopic methods. The compounds were
structurally characterized by X-ray diffraction studies. Additionally, electrochemical,
2

luminescence, antioxidant and anticancer properties of the compounds were investigated. The
electrochemical and photoluminescence properties of the imine compounds were investigated
in different solvents and concentrations.
2. Experimental
2.1 Materials and measurements
All reagents and solvents were of reagent-grade quality and obtained from commercial
suppliers (Aldrich or Merck). Elemental analyses (C,H,N) were performed using a LECO
CHNS 932. Infrared spectra were obtained using KBr disc (4000-400 cm-1) on a Perkin
Elmer Spectrum 100 FT-IR. The electronic spectra in the 200–900 nm range were obtained on
a Perkin Elmer Lambda 45 spectrophotometer. Mass spectra of the ligands were recorded on a
1
LC/MS APCI AGILENT 1100 MSD spectrophotometer. H and 13C NMR spectra were
recorded on a Bruker 400 MHz instrument and TMS was used as an internal standard and
DMSO-d6 as solvent. The thermal studies of the compounds were performed on a Perkin
Elmer STA 6000 simultaneous Thermal Analyzer under nitrogen atmosphere at a heating rate
of 10 °C/min.
The single-photon fluorescence spectra of the Schiff base compounds L1 and L2 were
collected on a Perkin Elmer LS55 luminescence spectrometer. All samples were prepared in
spectrophotometric grade solvents and analysed in a 1 cm optical path quartz cuvette. The
solutions of ligands (1.0 x 10-3-1.0 x 10-7 mol L-1) were prepared in DMF solvent. To
investigate the solvent effect on the photoluminescence spectra of the ligands, the DMF,
DMSO, CHCl3, CH3OH and C2H5OH solutions (1.0 x 10-3 mol L-1) of the compounds were
used.
A stock solution of concentrations of 1.0 x 10-3 M and 1.0 x 10-4 M of Schiff base
compounds was prepared in DMF for electrochemical studies. Cyclic voltammograms were
recorded on a Iviumstat Electrochemical workstation equipped with a low current module
(BAS PA–1) recorder. The electrochemical cell was equipped with a BAS glassy carbon
working electrode (area 4,6 mm2), a platinum coil auxiliary electrode and a Ag+/AgCl
reference electrode filled with tetrabutylammonium tetrafloroborate (0.1 M) in DMF and
CH3CN solution and adjusted to 0.00 V vs SCE. Cyclic voltammetric measurements were
performed at room temperature in an undivided cell (BAS model C–3 cell stand) with a
platinum counter electrode and an Ag+/AgCl reference electrode (BAS). All potentials are
reported with respect to Ag+/AgCl. The solutions were deoxygenated by passing dry nitrogen
3

through the solution for 30 min prior to the experiments, and during the experiments the flow
was maintained over the solution. Digital simulations were performed using DigiSim 3.0 for
windows (BAS, Inc.). Experimental cyclic voltammograms used for the fitting process had
the background subtracted and were corrected electronically for ohmic drop. Mettler Toledo
MP 220 pH meters was used for the pH measurements using a combined electrode (glass
electrode reference electrode) with an accuracy of ±0.05 pH.
Data collection for X-ray crystallography was completed using a Bruker APEX2 CCD
diffractometer and data reduction was performed using Bruker SAINT [15]. SHELXTL was
used to solve and refine the structures [16].
2.2 Synthesis of the Schiff base compounds
The benzaldehyde derivatives (1 mmol; 136 mg 2-methoxy benzaldehyde for L1 or 166 mg
3,4-dimethoxy benzaldehyde for L2) in ethanol (20 mL, absolute) and 4-aminophenol
(1 mmol, 109 mg) in ethanol (20 mL, absolute) were mixed and refluxed for about 10 h at
80 °C. The color of the solution changed to yellow. After cooling the solution, the resulting
precipitate was filtered and washed with cold ethanol. Single crystals of the Schiff base
compounds (L1 and L2) suitable for X-ray diffraction study were obtained by slow
evaporation of the compounds in ethanol. Physical properties and other spectroscopic data are
given below.
L1: (C14H13NO2). Yield: 90%, color: yellow, m.p.: 160 °C. Elemental analyses, found (calcd.
%): C, 74.03 (73.99); H, 5.80 (5.77); N, 6.20 (6.16). 1H NMR (DMSO-d6, δ (ppm)): 8.64 (s,
CH=N, 1H), 7.90-6.30 (m, Ar-H, 8H), 3.80 (s, OCH3, 3H). 13C NMR (DMSO-d6, δ (ppm)):
164.30 (CH=N), 158.20-106.60 (Ar-C), 57.15 (OCH3). Mass spectrum (LC/MS
APCI): m/z 228 [M+1]+ (100%), m/z 229 [M + 2]+ (22%), m/z 213 [M –CH3]+ (5%). FT-IR:
(KBr, cm-1): 3200 ν(O-H), 3010 ν(C-H)aromatic, 2960 ν(C-H)alph, 1605 ν(CH=N).
L2: (C15H15NO3). Yield: 85%, color: yellow, m.p.: 155 °C. Elemental analyses, found (calcd.
%): C, 70.05 (70.02); H, 5.90 (5.88); N, 5.40 (5.44). 1H NMR (DMSO-d6, δ (ppm)): 9.42 (s,
OH, 1H), 8.72 (s, CH=N, 1H), 7.95-6.62 (m, Ar-H, 7H), 3.84, 3.88 (s, OCH3, 6H). 13C NMR
(DMSO-d6, δ (ppm)): 163.62 (CH=N), 160.87-98.54 (Ar-C), 56.25, 55.93 (OCH3). Mass
spectrum (LC/MS APCI): m/z 258 [M+1]+ (100%), m/z 259 [M + 2]+ (23%), m/z 120
[C7H6NO]+ (48%). FT-IR: (KBr, cm-1): 3077 ν(O-H), 3014 ν(C-H)aromatic, 2947 ν(C-H)alph
,
1616 ν(CH=N).
4

2.3 X-ray structure solution and refinement for the compounds L1 and L2
Data for the compounds L1 and L2 were collected at 150(2)Kº on a Bruker Apex2 CCD
diffractometer using Mo-Kα radiation (λ = 0.71073Å). The structures were solved by direct
methods and refined on F2 using all the reflections [15]. All the non-hydrogen atoms were
refined using anisotropic atomic displacement parameters and hydrogen atoms bonded to
carbon atoms were inserted at calculated positions using a riding model. Hydrogen atoms
bonded to oxygen atoms in were located from difference maps and refined with temperature
factors riding on the carrier atom. Details of the crystal data and refinement are given Table 1.
2.4 Anticancer activity studies of the Schiff base compounds
2.4.1 Preparation of samples
Stock solutions of the samples were prepared in DMSO and diluted with Dulbecco’s modified
eagle medium (DMEM). DMSO final concentration is below 1% in all tests.
2.4.2 Cell lines and cell culture
HeLa cancer cell lines were grown in Dulbecco’s modified eagle medium (DMEM)
supplemented with 10% fetal bovine serum (FBS), 2% penicilin streptomycin. The medium
was changed twice a week.
2.4.3 Cell proliferation assay
The Real Time Cell Analyzer-Single Plate (RTCA-SP, xCELLigence) instrument (Roche
Applied Science, Basel, Switzerland) was used to visualize the antiproliferative effects of the
compounds L1 and L2 on human cervical cancer (HeLa) cells. RTCA-SP is a combination of
four parts: an E-Plate 96, a Single Plate (SP) station that is kept in an incubator and holds the
E-Plate 96, an analyzer and a computer with RTCA software. The E-Plate 96 wells have an
inner volume of 243±5 ꢀL and their bottoms are coated with golden electrodes. The
xCELLigence measures impedance differences set by an operator in order to derive cell index
values at time points and impedance differences as well as the cell index values depends on
the cell activity at the bottom of the wells. The higher cell index value depends on the higher
the cell population growing at the bottom and the greater the spreading of the cells. RTCA
allows researchers to analyze cell behavior in a labeling-free cell-based assay and produces a
real-time profile of the cells.
5

The HeLa cell line was cultured in DMEM-HG supplemented with 100 mL L−1 heat-
inactivated FBS and 20 mL L−1 penicillin/streptomycin at 37 C in a humidified atmosphere
∘
of 5% CO2.
To determine the antiproliferative effects of the synthesised imines, HeLa cells were first
detached from the tissue culture flask by the treatment with trypsin/ethylenediaminetetraacetic
acid solution. After detachment, the same volume of culture medium was added to this cell
suspension and gently mixed. Then the suspension was partitioned into Falcon tubes and
centrifuged. Meanwhile, 50 ꢀL of culture medium was added to each well of the E-Plate 96
and left in the hood for 15 min and in the incubator for 15 min to allow the electrodes to
equilibrate with the culture medium. After this period, the E-Plate 96 was inserted into the
RTCA-SP station and a background measurement was performed. Immediately afterwards,
the E-Plate 96 was ejected from the station and 100 ꢀL of cell suspension adjusted to a
concentration of 2.5 × 104 HeLa cells per 100 ꢀL−1 was added to each well. Three wells were
left blank to check if there would be an increase due to the culture medium. The plate was left
in the hood for another 30 min for the cells to adhere to the bottom, after which the plate was
inserted into the RTCA-SP station and a measurement lasting 80 min was started. After this
period, the plate was ejected from the station and plant extracts (in dimethyl sulfoxide
(DMSO)/culture medium; the final concentration of DMSO in the wells was less than 10 mL
L−1) were added to the wells at three different concentrations (100, 50 and 10 ꢀg mL−1), and
the final volume of each well was adjusted to 200 ꢀL with culture medium. Then the plate
was connected to the station and a measurement lasting 48 h was started. The measurement
was made in triplicate.
2.4.4 Statistical analysis
All experiments were performed in triplicate. The proliferation tests were evaluated as
mean±SD of three measurements of cell index values by ANOVA at 1% significance level
and compared by Tukey’s one-way comparison test for differences using Minitab 14 (Minitab
Inc., State College, PA, USA).
6

3. Results and Discussion
In this manuscript, two imine compounds L1 and L2 containing methoxy groups were
prepared. These compounds are stable at room temperature without decomposing. They are
soluble in polar organic solvents such as, EtOH, MeOH, CHCl3, DMF, DMSO etc. and
partially soluble in apolar solvents such as, hexane, heptane, toluene etc. The compounds
were characterised by the spectroscopic and analytic techniques. Structural characterizations
of the synthesised compounds were determined by single crystal X-ray diffraction technique.
The proposed structures of the imine compounds are shown in Fig. 1. The imine compounds
were purified by the crystallization method. Because, if their purification is performed by the
colon technique, the compounds may be decompose as hydrolysis. In such cases, it is better to
purify the Schiff bases by re-crystallization. A problem in studying with the Schiff bases of
the aromatic hydroxycarbonyls derives essentially, even in neutral solutions several of species
may be appear. Additionally to enolimines, hemiacetals and their hydrolysis products,
tautomeric species, may also formed, like keto-amines or cyclic diamines formed from mono
Schiff bases of diamines. This truth is often omitted, although it must be taken account of
when protonation equlibria are analysed [17].
1
In order to clarify the structures of the Schiff bases (L1 and L2), their H(13C)-NMR
spectra were investigated and obtained data are given in the experimental section. The
1
1H(13C)-NMR spectra of the imine compound L2 are given in Figs. 2a-d. In the H-NMR
spectrum of the imine compound L2, the broad band at 9.42 ppm can be attributed to the
1
hydrogen atom of the OH group. In the H-NMR spectra of the imine compounds L1 and L2,
the singlets at 8.64 and 8.72 ppm can be attributed to the protons of the azomethine groups.
The aromatic ring protons are shown in the 7.95-6.30 ppm range as multiplet. The signals of
the methoxy groups on the benzene rings are shown in the 3.88-3.80 ppm range as singlet
13
signals. In their C-NMR spectra, the signals at 164.30 and 163.62 ppm may be assigned to
the carbon atom of the azomethine group. Aromatic carbon atoms are shown in the 160.87-
98.54 ppm range. The signals in the 57.15-55.93 ppm range can be attributed to the methoxy
carbon atoms.
The infrared spectral data of the compounds are given in the experimental section. In
the FT-IR spectra of the imine compounds (L1 and L2), the broad bands in the 3200 and 3070
cm-1 may be assigned to the ν(O-H) vibrations. The vibration signals at 3010 and 3014 cm-1
can be attributed to the aromatic C-H strechings. The bands at 2960 and 2947 cm-1 may be
assigned to the ν(C-H)alph vibrations. The azomethine group [ν(CH=N)] vibrations are shown
at 1605 and 1616 cm-1.
7

The most useful techniques for investigating the tautomeric forms (Fig. 3) of the
ligands are UV and NMR spectroscopies, whereas IR seems to be of limited value here
because the locations of the ν(C-O) and ν(C=O) stretching frequencies in the spectra are
obscured by the abundance of aromatic skeletal modes. The CH=N and OH groups of the
enolimine–ketoamine tautomers are capable of forming hydrogen bonds with suitable
solvents. Due to the stronger hydrogen bond donor ability of the OH groups compared with
that of the CH=N group, the formation of hydrogen bonds mainly with chloroform and
ethanol solvents should stabilize the enol-imine form, whereas the basic ketoamine form
should be more stabilized in solvents such as toluene and heptane. It has been shown by
electronic spectroscopy that the equilibrium in less polar and non-polar solvents is shifted
towards the ketoamine form. On the other hand, hydrogen bond acceptor solvents favour the
enolimine form due to hydrogen bonding [18]. In order to investigate the solvent effect, the
spectra of the compounds were taken in the polar and apolar organic solvents. These solvents
are MeOH, EtOH, DMF, DMSO CH2Cl2, CHCl3, n-hexane, n-heptane, cyclohexane and
tolüene solutions. The electronic spectra of the compounds L1 and L2 in the 1 x 10-3 M
concentration and in the different solvents are shown in Figs. 4a-d. In addition, the obtained
data from the electronic absorption studies are given in the Table 1. The imine compounds
show different tautomeric forms depending on the solvent polarity. In polar organic solvents,
the imine compounds show the bands in the 371-291 nm range and this bands may be
attributed to the n-π* transitions [19]. In this region, the compounds have the bands with
maximum intensity in the methanol solution. The bands with maximum intensity in the 208-
273 nm range can be assigned to the benzenoid transitions (π-π* and δ-δ*). In the non-polar
organic solvents, the n-π* transitions of the imine compounds are shown in the 515-292 nm
range. The n-π* transition of the imine compound L1 shifted to the longer wavelength in the
n-heptane solution. The π-π* and δ-δ* transitions were shown in the 289-212 nm range.
Single crystals suitable for single crystal X-ray diffraction study were obtained from
slow evaporation of the ethanol solution of the imine compounds L1 and L2. Molecular
structures of the imines L1 and L2 are shown in Figs. 5 and 6. The molecular structures of two
imine compounds are broadly similar, differing principally in the position, the number of
methoxy (-OCH3) groups and dihedral angles between aromatic rings. While the compound
L1 has only one methoxy group located on the o-position with respect to the imine bond
(C=N), the compound L2 contains two methoxy groups on the m-p-positions with respect to
the imine bond. For the imine compounds L1 and L2, all bond lengths and angles in the phenyl
8

rings have normal Csp2–Csp2 values and are in the expected ranges. The crystallograhic data
for the imine compounds L1 and L2 are given in Table 2. The C=N distances in both
compounds are within a typical C=N double bond character. The compounds show
intermolecular phenol-imine hydrogen bonds. Hydrogen bond parameters for the imines L1
and L2 are given in Tables 3 and 4, respectively.
In the structure of the imine L1, the asymmetric unit contains two independent Schiff
base molecules (molecule A and B) differing in the dihedral angles between two aromatic
rings. The dihedral angles between two aromatic rings are 46.74(4) and 7.92(5)º for molecules
A and B, respectively. In the structure of the imine L1, four molecules are assembled by
intermolecular hydrogen bonds forming a supramolecular 28-membered ring (Fig. 7).
Molecular packing of the imine L1 is determined by OH····N and C-H····O hydrogen
bondings (Table 3). Packing diagram of L1 is shown in Fig. 8.
The asymmetric unit of L2 contains four independent imine molecules (A-D). All four
molecules exhibit similar bond length and angles, however, the dihedral angle between two
aromatic rings in each molecule is different from each other. The dihedral angles between
aromatic rings in four molecules (A-D) of the imine L2 are 21.34(9), 10.03(9), 71.94(6) and
38.04(8)º for molecules (A-D), respectively. Overlay of four different conformers
emphasising the twist in the phenol ring is shown in Fig. 9. The molecules of the imine L2 are
connected via phenol-imine (OH····N) intermolecular hydrogen bonding resulting in a
hydrogen bonded chain Fig. 10.
3.1 The effect of different solvent and concentration on the photoluminescence properties of
the imine compounds L1 and L2
In order to determine the effect of the solvent polarity, the photoluminescence
properties of the imine compounds L1 and L2 were investigated in the DMF, DMSO, CHCl3,
CH3OH and C2H5OH solvents. The emission and excitation spectra of the Schiff base
compounds L1 and L2 in the CHCl3 and C2H5OH solvents are shown in Figs. 11a-d and the
obtained data are given in Tables 5 and 6. Dielectric constant order of the solvents is
DMSO>DMF>CH3OH>C2H5OH>CHCl3. In the excitation spectra of the imine compound L1
in the DMF and DMSO solvents, two bands were shown in the 320-476 nm range. On the
other hand, in the CHCl3, CH3OH and C2H5OH solvents, only one band was shown in the
395-431 nm range. While the excitation band shifted to the shorter wavelenght in DMSO
solution, the band shifted to the longer wavelenght value in DMF solution. In the excitation
spectra of the imine compound L2, the compund has two excitation bands in the 245-400 nm
9

range in the CHCl3 and CH3OH solvents. In the C2H5OH solvent, the compound L2 has three
excitation bands in the 239-363 nm range. In the DMF and DMSO solutions, only one band
was shown in the 407-417 nm range. The excitation bands of the imine compound L1 shifted
to the longer wavelenghts compared to the imine compound L2.
The emission spectra of the compounds L1 and L2 show two or three emission bands
in the 619–832 nm range in the DMF, DMSO, CHCl3, CH3OH and C2H5OH solutions. The
emission peaks of the compound L1 shifted to the longer wavelengths. The
photoluminescence emission peaks of the imine compounds apparently produce red shift with
the introduction of the electron donating groups. The introduction of the electron donating
groups by mesomeric and inductive effects causes the fluorescence characteristic emission
peaks of the imine compounds to the slightly red shift. The reason is that the electron density
of the phenyl ring is increased with the δ-π hyper conjugation effect. The Schiff bases with
methoxy substituents possess p-π conjugation that can increase their photoluminescence
emission intensity. As the imine compound L2 have p-methoxy substitute group, it shows a
good conjugation and rigid planar structure. It may be that the extended π-conjugation would
induce an excited state resonance contribution of the methoxy groups to the benzene rings in
the increased polarity.
In order to investigate the effects of the concentration on the photoluminescence
properties of the imine compounds L1 and L2, the solutions in 1.0x10-3-1.0x10-7 M range in
DMF, DMSO, CHCl3, CH3OH and C2H5OH solutions were used. In the spectra of the
compounds, the emission peaks did not shift to the shorter or longer wavelenghts (SW) from
1.0x10-3 M to 1.0x10-7 M concentration. However, the intensity of the absorption bands were
decreased towards to lower concentrations. This situation occur due to the lesser substance
quantity in lower concentrations. Similar properties were also shown for the excitation peaks.
3.2 The electrochemical properties of the compounds L1 and L2
Electrochemical properties of the imine compounds (L1 and L2) were studied in
CH3CN and DMF - 0.1 M NBu4BF4 as supporting electrolyte at 298 K. In order to study the
effects of the solution concentration and the scan rates, two different concentrations (1.0x10-3
and 1.0x10-4 M) and the scan rates (100, 250, 500, 750 and 1000 mV/s) were used against an
internal ferrocence-ferrocenium standard. The obtained data are given in Table 7. The
electrochemical curves of the compounds L1 and L2 in DMF and CH3CN solutions are shown
in Figs. 12a-d.
10

In the 1.0x10-3 M CH3CN solution, the imine compound L1 shows three anodic peak
potentials in the -0.79-1.13 V range at all scan rates. In the same solution, the compound has
two cathodic peaks in the -0.09-0.74 V range. In DMF solution, while the compound has two
anodic peaks in the -0.85-0.65 V range, the compound has three cathodic peaks in the -0.94-
0.89 V range. The anodic peaks in the 1.09-1.13 V range in the CH3CN solution of the
compound L1 were disappeared in DMF solution. On the other hand, the cathodic peaks in the
-0.90-0.94 V range were determined. In the 1x10-4 M CH3CN solution, the compound L1 has
two anodic peaks in the -0.49-1.15 V range. But, this compound has three cathodic peaks in
the -1.06-0.61 V range. In DMF solution, the compound L1 has two anodic and cathodic peaks
in the -0.58-0.30 and -0.79-0.18 V range, respectively. From the obtained data for the imine
compound, the scanning rate, solvent polarity and solution concentration were effective on the
redox processes. At all scan rates, the redox processes for the imine compound L1 are either
quasi-reversible or irreversible events (Fig. 13a).
In the 1.0x10-3 M CH3CN solution, the imine compound L2 shows three irreversible
anodic peak potentials in the -0.64-1.15 V range at all scan rates. But, the imine has two
irreversible cathodic redox processes in the -1.16-0.55 V range. In DMF solution, the imine
L2 has three piece anodic and cathodic peaks in the -0.76-0.67 and -0.76-0.80 V range,
respectively. At all scan rates, the redox processes are reversible (Fig. 13b). On the other
hand, in the 1x10-4 M CH3CN solution, the imine compound L2 has three irreversible anodic
and two cathodic peaks in the -0.56-1.09 and -0.15-0.51 V range, respectively. As distinct
from this, in DMF solution, the imine compound L2 has reversible three anodic and cathodic
redox potentials in the -0.49-1.27 and -0.69-1.0 V range, respectively. In the reversible redox
process, the Schiff base compounds have been converted to the keto-amine forms [20]. In this
process, the oxygen atoms of the methoxy groups of the organic compounds transfer the
electrons to the benzenoid rings and then to the nitrogen atoms by the resonance. This process
occurs as the reversible. While the compound L1 has only one methoxy group (ortho position
on the benzene ring), the other compound L2 have two methoxy groups (meta and para
positions on the benzene ring). Although the methoxy groups decrease the electron density of
the benzenoid rings by the inductive effect, the electron density increase by the mesomeric
effect. Electron donating groups to the benzene rings shift the potentials from the positive to
negative regions. As a result, the obtained anodic and cathodic data in the CH3CN shifted to
the more negative regions than in DMF solution.
11

3.3 The antiproliferative activities of compounds L1 and L2
Figure 11a-b shows the results of xCELLigence real-time monitoring of the proliferation of
HeLa cells treated with L1 and L2. The cell index measurements indicated that the anticancer
activities of synthesised imine derivatives are similar (Figs. 14a,b) except for slight
differences in concentrations 50 and 100 µM which are higher activities. The antiproliferative
activities of the compounds L1 and L2 were very weak to compare with concentrations of 10
µM with 50 and 100 µM. However, these compounds have remarkable activities aganist HeLa
cell line in between 10 and 50 µM. According to the recent study, where a new Schiff base
(N-3,4-dihydroxybenzylidene) ferroceneamine showed an excellent biological activity against
the HeLa cancer cell line [21], the hydroxy groups play an important role in anticancer
activitiy [22]. The hydroxy and methoxy-substituted Schiff bases have received considerable
attention due to their potential anticancer activity. While the compound L1 has one methoxy
group on the ortho position of the benzenoid ring, the other compound L2 has two methoxy
groups on the meta and para position of the benzenoid ring. The methoxy groups give the
electron to the aromatic rings by the mesomeric effect. But, the compound L1 has the higher
antiproliferative activity than the L2. On the other hand, the compounds have only one imine
group and this group has an impact on anticancer activity. This showed that different
structural requirements exhibit selectivity against different cancer cell lines. In addition, the
activity of the compounds L1 and L2 have increased in dose-depended manner from 10 to 50
µM. The antiproliferative activity of compound L1 was more effective than the compound L2
at 100 and 50 µM aganist HeLa cell line. In the light of the results, it can be suggested that the
compounds L1 and L2 could be developed as an anticancer drug.
Conclusion
In this work, we synthesized two imine compounds L1 and L2 containing methoxy groups and
characterized by the analytical, spectroscopic and X-ray techniques. The photoluminescence
and electrochemical properties of the compounds were investigated. The compounds L1 and
L2 show the emission bands at the higher wavelenghts. In addition, the compounds indicate
three emission bands. In the electrochemical studies, the compounds showed reversible redox
processes at the some scan rates. The molecular structures of two imine compounds are
broadly similar, differing principally in the position, the number of methoxy (-OCH3) groups
and dihedral angles between aromatic rings. While the compound L1 has only one methoxy
group located on the o-position with respect to the imine bond (C=N), the compound L2
12

contains two methoxy groups on the m-p-positions with respect to the imine bond. The
compounds L2 and L1 showed higher anticancer activities at higher concentrations. This
situation may be due to methoxy groups on the benzenoid rings of the imine compounds.
Supplementary Information
CCDC numbers 1034582 and 1034583 contain the supplementary crystallographic data for L1
and L2, respectively. Bond lenghts and angles of the compounds were given in Supplementary
Information.
These
data
can
be
obtained
free
of
charge
via
www.ccdc.cam.ac.uk/data_request/cif, by e-mailing data_request@ccdc.cam.ac.uk, or by
contacting The Cambridge Crystallographic Data Centre 12 Union Road Cambridge CB2
1EZ, UK Fax: +44(0)1223-336033.
Acknowledgments: We are grateful to The Scientific & Technological Research Council of
Turkey (TUBITAK) (Project number:109T071) for the support of this research. The authors
are also grateful to Prof. Dr. Vickie McKee and the Department of Chemistry, Loughborough
University for X-ray data collection.
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14

Figure captions
Fig. 1. Proposed structures of the synthesized imine compounds.
Fig. 2a-d. 1H(13C)-NMR (a) , HMBC (b), HETCOR (c) and TOSCY (d) spectra of the imine
compound L2.
Fig. 3. Tautomeric forms of the imine compounds L1 (a) and L2 (b) in the polar and non-polar
organic solvents.
Figs. 4a-d. Absorption spectra of the imine compounds L1 and L2 in the polar and non-polar
organic solvents.
Fig. 5. Molecular structure of the compound L1 with atom numbering. Intra-molecular
hydrogen bonding (OH····N) is shown as dashed lines.
Fig. 6. Molecular structure of the compound L2. Hydrogen atoms are omitted for clarity.
Intra-molecular hydrogen bonds (OH····N) are shown as dashed lines.
Fig. 7. Hydrogen bonded 28 membered macrocyclic in the imine L1, hydrogen atoms bonded
to carbon atoms are not shown for clarity.
Fig. 8. Packing plot of the imine L1 viewing down c axis, hydrogen bonds are shown as dash
lines and hydrogen atoms are omitted for clarity.
Fig. 9. Overlay of four independent molecules in the imine L2, emphasising the twist in the
phenol ring. Hydrogen atoms are omitted for clarity.
Fig. 10. Packing diagram of the imine L2 showing hydrogen bond chain. Hydrogen atoms are
omitted for clarity.
Figs. 11a-d. Photoluminescence (excitation and emission) spectra of the imine compounds L1
and L2 in different concentration and solvents.
Figs. 12a-d. CV curves of the imine compounds L1 and L2 in different concentrations and
solvents in the 100-1000 mV/s range.
Figs. 13a,b. The proposed redox mechanism for the imine compounds L1 (a) and L2 (b) in the
solution media.
Figs. 14a,b. Anticancer activities of the imine compounds L1 and L2.
15

O
O
O
N
N
OH
OH
L1
L2
Fig. 1. The proposed structures of the synthesized imine compounds L1 and L2.
24

Figure(s)
9.5
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
ppm
170
160
150
140
130
120
110
100
90
80
70
60
50
40 ppm
a) 1H(13C)-NMR
25

26

27

1
Figs. 2a-d. H(13C)-NMR (a) , HMBC (b), HETCOR (c) and TOSCY (d) spectra of the imine compound L2.
28

Figure(s)
O+
O
O
N
O
N
NH
OH
OH
keto-imine
keto-amine
enol-imine
a)
O+
O
O
O
O
O
N
NH
N
O
OH
OH
keto-imine
keto-amine
enol-imine
b)
Fig. 3. Tautomeric forms of the imine compounds L1 (a) and L2 (b) in the polar and non-polar
organic solvents.
29

a) L1
30

b) L1
31

c) L2
32

d) L2
Figs. 4a-d. Absorption spectra of the imine compounds L1 and L2 in the polar and non-polar organic solvents.
33

Figure(s)
Fig. 5. Molecular structure of the compound L1 with atom numbering. Intra-molecular
hydrogen bonding (OH····N) is shown as dashed lines.
34

Figure(s)
Fig. 6. Molecular structure of the compound L2. Hydrogen atoms are omitted for clarity.
Intra-molecular hydrogen bonds (OH····N) are shown as dashed lines.
35

Figure(s)
Fig. 7. Hydrogen bonded 28 membered macrocyclic in the imine L1, hydrogen atoms bonded
to carbon atoms are not shown for clarity.
36

Figure(s)
Fig. 8. Packing plot of the imine L1 viewing down c axis, hydrogen bonds are shown as dash
lines and hydrogen atoms are omitted for clarity.
37